We want to understand the structural origins of functional diversity in the (beta/alpha)8 barrel fold, the most commonly observed fold in enzymes. We have identified a group of homologous (beta/alpha)8-barrel fold enzymes that catalyze reactions with unrelated substrates and mechanisms in different metabolic pathways. Orotidine 5'-phosphate decarboxylase (OMPDC) is the best characterized member of this "suprafamily." We propose to determine structure/function relationships for other members of the OMPDC suprafamily so that we can understand the structural strategies for using a homologous scaffold to catalyze unrelated reactions. These studies significantly expand the scope of functional diversity beyond those found in mechanistically diverse enzyme superfamilies, e.g., the enolase and crotonase superfamilies, in which homologous enzymes catalyze different reactions that share a common partial reaction. The four Specific Aims integrate mechanistic and structural studies: the mechanistic studies will be performed in Dr. Gerlt's laboratory at Illinois (P.I.); the structural studies will be performed in Dr. Rayment's laboratory at Wisconsin (Co-P.I.): 1) Structure/function relationships will be established for 3-keto-L-gulonate 6-phosphate decarboxylase that catalyzes Mg2+-dependent decarboxylation of beta-ketoacids via an enediolate intermediate. 2) Structure/function relationships will be established for D-arabino-hex-3-ulose 6-phosphate synthase that catalyzes Mg2+-dependent aldol condensation via an enediolate intermediate. 3) Structure/function relationships will be established for D-ribulose 5-phosphate 3-epimerase that catalyzes divalent metal-independent 1,1-proton transfer via an enediolate intermediate. 4) A structural blueprint for functional diversity in the ((beta/alpha)8-barrel fold will be tested.